The malaria parasite sporozoite stage is the only known invasive form of a living organism that migrates through and ultimately invades different tissues in vertebrate and invertebrate hosts. During these intriguing journeys in two different hosts, the sporozoite numbers drastically decline and only very few sporozoites reach and invade hepatocytes. Therefore, sporozoites are considered excellent targets for malaria intervention strategies. Despite several attempts to identify the transcript abundance among spatially and temporally different sporozoite populations, the mechanisms underlying the sporozoite success in gaining motility and switching infectivity are still unknown. A very distinctive feature of Plasmodium sporozoites is the heterogeneity in maturation, egress and invasion of salivary glands. This means that sporozoites mature and egress from oocysts over a period of several days following their morphogenesis in oocysts. A clear example of the heterogeneity in sporozoites is the fact that oocyst sporozoites (ooSpz), when injected in high numbers, can invade and develop inside hepatocytes in mammals, despite of being programmed to invade mosquito salivary glands. Therefore, heterogeneity will be always a major obstacle in trying to identify any common mechanisms or pathways of infectivity or motility phenotypes in sporozoites. Herein, we applied a serial selection pressure to select for the Liver-Infectious-Oocyst-Sporozoites (LIOS). The selection pressure was applied by bypassing the obligatory salivary gland invasion step and injecting ooSpz into susceptible mice, and repeating these steps for the resulting LIOS induced blood stage parasites. Our preliminary results showed that after five passages of LIOS through mice, they became 1000 fold more infectious than wild-type (WT) ooSpz. Based on these findings, we hypothesize that infectivity, motility and maturation determinants and regulatory elements could be identified from homogenous sporozoite populations that are phenotypically distinct. In specific aim 1, we are proposing to generate HI-LIOS (highly Infectious-LIOS) that are as highly infectious as sgSpz to mammals, in GFP background (HI-LIOS-GFP). We will select for phenotypically distinct clones (liver infectious and motile vs. liver infectious but not motile) using FACS sorting and infectivity-dextran assays. Detailed phenotypic analysis will be conducted to confirm and analyze all phenotypes displayed by HI- LIOS-GFP vs. their original WT-GFP clones. In specific aim 2, we are proposing to compare selected HI-LIOS-GFP to WT-GFP clones at days 10, 15 and 18 post mosquito infections by genome and transcriptome analyses. We will purify sporozoites by FACS sorting from three mosquito tissues (midgut, hemolymph and salivary glands) to isolate RNA and DNA at all three time-points. This will help to reveal initial insights about the distinct phenotypic differences between LIOS-GFP clones and between LIOS-GFP and WT-GFP clones. Further epigenetic and proteome analyses, along with confirmation of the genome wide data, will be planned in future proposals and studies with LIOS, as these tools that will enable us for the first time to homogenize sporozoite populations to dissect out their functions. The outcome of this project may help in identifying new gene expression regulation pathways for Plasmodium and novel intervention targets for malaria eradication.